An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

Fluorescence photobleaching recovery in the confocal scanning light microscope

Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three-dimensional (3-D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)-FPR experimental data. The 2-D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well-defined volume element in bulk samples. Two-dimensional diffusion coefficients of 45-nm latex spheres, of FITC molecules and of a 2·45-nm protein-FITC complex in water-glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10^?6 cm²/s can be measured.     

Confocal scanning light microscopy with high aperture immersion lenses

The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.     

Theoretical versus experimental resolution in optical microscopy

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.     

Reconstruction and display of curvilinear objects from optical section data using 3-D curve fitting algorithms

Biological objects resembling filaments are often highly elongated while presenting a small cross-sectional area. Examination of such objects requires acquisition of images from regions large enough to contain entire objects, but at sufficiently high resolution to resolve individual filaments. These requirements complicate the application of conventional optical sectioning and volume reconstruction techniques. For example, objective lenses used to acquire images of entire filaments or filament networks may lack sufficient depth ( Z ) resolution to localize filament cross-sections along the optical axis. Because volume reconstruction techniques consider only the information represented by a single volume element (voxel), views of filament networks reconstructed from images obtained at low Z -resolution will not accurately represent filament morphology. A possible solution to these problems is to simultaneously utilize all available information on the path of an object by fitting 3-D curves through data points localized in 2-D images. Here, we present an application of this approach to the reconstruction of microtubule networks from 2-D optical sections obtained using confocal microscopy, and to synthesized curves which have been distorted using a simple mathematical model of optical sectioning artefacts. Our results demonstrate that this strategy can produce high resolution 3-D views of filamentous objects from a small number of optical sections.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

A theory for multiphoton fluorescence imaging in high aperture scanning optical microscopes employing finite sized detectors is presented. The effect of polarisation of the fluorescent emission on the imaging properties of such microscopes is investigated. The lateral and axial resolutions are calculated for one-, two- and three-photon excitation of p-quaterphenyl for high and low aperture optical systems. Significant improvement in lateral resolution is found to be achieved by employing a confocal pinhole. This improvement increases with the order of the multiphoton process. Simultaneously, it is found that, when the size of the pinhole is reduced to achieve the best possible resolution, the signal-to-noise ratio is not degraded by more than 30%. The degree of optical sectioning achieved is found to improve dramatically with the use of confocal detection. For two- and three-photon excitation axial full width half-maximum improvement of 30% is predicted.     

Three-dimensional visualization methods for confocal microscopy

Three-dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three-dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi-transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.     

Optical sectioning with a fluorescence confocal SLM: procedures for determination of the 2-D digital modulation transfer function and for 3-D reconstruction by tessellation

We have determined the operational parameters of a confocal scanning laser microscope (CSLM) used for fluorescence imaging. The system performance was characterized by the modulation transfer function (MTF), calculated from an edge response function (ERF) corresponding to a standard test pattern overlaying fluorescence solutions on a microscopic slide. Signal truncation error was avoided by making the ERF continuous at its limits through superposition of a suitable linear curve. We determined an appropriate scanning step density by defining a compromise between the requirements of the sampling theorem with respect to aliasing and the need for minimal suppression of higher spatial frequencies. These procedures for choosing the sampling density of the total digital CSLM permit a systematic optimization of the image acquisition parameters. A reproducible digital image was obtained, an important prerequisite for subsequent 3-D image reconstruction. The latter was accomplished by first developing and applying a maximally automated algorithm for finding closed and distinct contours (corresponding to objects in the 2-D optical sections). The missing information between contour loops was then interpolated by tessellation (triangulation) using a minimal polygon edge length criterion capable of describing closed surfaces even for adjacent contours with highly dissimilar geometries. In this procedure, we define an average shift length which compensates for the inherent disadvantage of run length encoding algorithms applied to different starting points in successive planes. The surface segments were used to calculate 3-D representations by applying a shading model, in the present applications specifically to chromosome I and the nucleolus of polytenized Chironomus thummi salivary gland nuclei.     

Resolution in light microscopy studied by computer simulation

Microscopic resolution can be characterized by K = x NA/δ, where x is the distance between two objects, or the interval of a grating, just resolved with light of wavelength λ and an objective of aperture NA. Using a computer simulation of imaging the following K values were obtained on the Sparrow resolution criterion for line and grating objects and various imaging methods (figures for the Rayleigh criterion, which assumes a finite contrast-sensitivity of the light detector, are in parentheses). Several results appear to be novel. Due to limitations discussed in the text some data are only approximate. With a grating object K is 1.0 (1.0) for axial coherent illumination, 0.5 (0.5) for coherent illumination at an obliquity NA_obi which just enters the objective aperture, 0.5 (about 0.53) for incoherent illumination, 0.5 (about 0.52) for illumination with a condenser aperture NA_c equal to NA, 0.5 (about 0.515) for transmitted-light confocal scanning, and 0.25 (about 0.38) for fluorescent confocal scanning. If the object consists of two parallel lines K is about 0.68 (0.71) for axial coherent illumination, about 0.44 (0.5) for incoherent illumination, 0.375 (about 0.48) for optimal partially coherent illumination in which NA_c may exceed NA, 0.44 (0.48) for transmitted-light confocal scanning, and 0.32 (0.41) for fluorescent confocal scanning. For inter-object distances of 1, 1.5 and 2 wavelengths, respectively, NA_c values of about 0.69, 0.5 and 0.375 gave optimal contrast and resolution irrespective of NA. The practice of setting NA_c to about two-thirds of the NA of a high-power objective is supported by the fact that a condenser aperture of about 0.69 gives excellent or optimum resolution and contrast for most inter-object distances and objective apertures tested, although with some distances and apertures reducing NA_c improved contrast slightly. The rule (sometimes attributed to Abbe) that resolving power is proportional to the mean of NA and NA_c is correct for oblique coherent illumination in the case of a grating object, provided NA_obl does not exceed NA. In the case of two isolated objects the rule is only approximately correct, but applies even if NA_obl is greater than NA. Coherent light at an obliquity of 0.5λ/x introduces a half-wavelength phase difference between two objects and permits their resolution (with perhaps an incorrect apparent inter-object distance) even with objective apertures approaching zero. In confocal scanning the width of the scanning spots has only a moderate effect on resolution, and two objects can sometimes be resolved with scanning spots wider than the inter-object distance provided the lens apertures are neither too small nor too large.     

Measurements of fibre direction in reinforced polymer composites

High-quality data on the three-dimensional (3-D) spatial distributions of glass and carbon fibres in fibre-reinforced polymer composites are important for both process control and the modelling of the mechanical and thermal properties of these composites. The advent of economical, high-speed, image analyser systems has enabled numerous research groups to measure directional distributions of fibre samples. Specimens are microtomed and polished and, using optical reflection microscopy, thousands of elliptical fibre images may be analysed within a short period of time. From the eccentricity of the fibre images, estimation of the angles (θ, φ) of each fibre relative to the vertical axis and within the measurement plane is deduced. However, this measurement is subject to considerable error. The confocal scanning laser microscope (CSLM), operating in fluorescence mode or reflection mode, is capable of improving the angular resolution (δθ, δφ) for all fibre directions. The ability of the CSLM to optically section glass and carbon fibre-reinforced polymer composites down to depths of 20 or 30 μm allows the user to determine accurate fibre directions from the apparent movement of fibre profiles. The CSLM has the potential for standardizing measurements of 3-D fibre directions in polymer composites and providing the quality directional data which are required for the theoretical modelling of composite processing and composite strength.     

Single-pinhole confocal imaging of sub-resolution sparse objects using experimental point spread function and image restoration

Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.     

Applications of confocal microscopy in industrial solid materials: some examples and a first evaluation

We report here the first results of the application of confocal scanning laser microscopy (CSLM) for the study of the microstructure of solid industrial materials. Glass-fibre-reinforced composites, heterogeneous and conductive polymers, homogeneous as well as heterogeneous catalyst (precursor) specimens and soils were examined. We conclude that both the fluorescence and reflection modes of CSLM can yield valuable information. In particular, the optical sectioning capability of CSLM appears to be of great value as it enables one to access the 3-D organization of the specimen without the need for a difficult and time-consuming specimen preparation procedure. However, local obscuration may be an important factor in confocal image formation, limiting the penetration capabilities of the technique for industrial materials.     

Precise 3D image alignment in micro-axial tomography

Micro (µ‐) axial tomography is a challenging technique in microscopy which improves quantitative imaging especially in cytogenetic applications by means of defined sample rotation under the microscope objective. The advantage of µ‐axial tomography is an effective improvement of the precision of distance measurements between point‐like objects. Under certain circumstances, the effective (3D) resolution can be improved by optimized acquisition depending on subsequent, multi‐perspective image recording of the same objects followed by reconstruction methods. This requires, however, a very precise alignment of the tilted views. We present a novel feature‐based image alignment method with a precision better than the full width at half maximum of the point spread function. The features are the positions (centres of gravity) of all fluorescent objects observed in the images (e.g. cell nuclei, fluorescent signals inside cell nuclei, fluorescent beads, etc.). Thus, real alignment precision depends on the localization precision of these objects. The method automatically determines the corresponding objects in subsequently tilted perspectives using a weighted bipartite graph. The optimum transformation function is computed in a least squares manner based on the coordinates of the centres of gravity of the matched objects. The theoretically feasible precision of the method was calculated using computer‐generated data and confirmed by tests on real image series obtained from data sets of 200 nm fluorescent nano‐particles. The advantages of the proposed algorithm are its speed and accuracy, which means that if enough objects are included, the real alignment precision is better than the axial localization precision of a single object. The alignment precision can be assessed directly from the algorithm's output. Thus, the method can be applied not only for image alignment and object matching in tilted view series in order to reconstruct (3D) images, but also to validate the experimental performance (e.g. mechanical precision of the tilting). In practice, the key application of the method is an improvement of the effective spatial (3D) resolution, because the well‐known spatial anisotropy in light microscopy can be overcome. This allows more precise distance measurements between point‐like objects.     

Second-order stereology for pores in translucent alumina studied by confocal scanning laser microscopy

The three-dimensional (3-D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3-D image of the pores. Computer-based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second-order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3-D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 μm.     

Image processing techniques for 3-D chromosome analysis

3-D karyotype analysis is developing rapidly due to the availability of confocal microscopes and CCD video cameras, and the development of 3-D processing techniques. Here, image enhancement and visualization techniques specifically designed for 3-D karyotype analysis are described. To facilitate a good comparison between the different techniques, the same 3-D image, obtained with a confocal scanning laser microscope (CSLM), of a mitotic prophase nucleus of a root-tip cell of Crepis capillaris was used throughout. Besides well-known stereoscopic presentation, another means of improving depth perception is shown, i.e. a solid modelling algorithm, which simulates the process of fluorescence. An interactive routine to dissect objects in the image is presented as an alternative for automated segmentation algorithms, which cannot be applied to closely apposed or merging objects. As an example of a convenient way to reduce the vast amount of data (2 Mbyte per image), a partly automated 3-D cursor is presented in detail. This cursor is used to trace the central axes of chromosomes and record them as strings of Cartesian coordinates. The advantages of a computer graphics display, which facilitates real-time rotation and hence is a powerful tool in studying 3-D features of chromosomes, are also shown.     

微型牙齿三维测量技术与系统

为方便扫描牙齿等微小物体的三维形貌,设计了一套专门用于口腔内部直接测量的牙齿三维扫描系统,该系统利用数字光栅投影仪投射结构光,通过远心成像镜头将光栅图像缩小,经光纤传像束传输投射到待测微小物体上,同时采用电子内窥镜获取图像,最后重构出物体的三维形貌。实验证明,该系统切实可行,满足牙齿扫描的微型化、直接化、灵活性的需求,拓展了结构光微测量三维形貌的适用范围。     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

Measurement-based evaluation of optical pathlength distributions reconstructed from simulated differential interference contrast images

Recently a method was presented for reconstructing optical pathlength distributions (OPDs) from images of weak phase objects obtained by a conventional differential interference contrast (DIC) microscope. A potential application of this technique is the determination of the mass of biological objects: by integrating the optical pathlength over the projected surface of the image of an object, a measure of the dry mass, i.e. the total mass of all solid constituents present in the object, is obtained. To assess the possibilities of DIC microscopy for this application, simulations were performed on computer-generated DIC images of objects of various sizes, shapes and orientation angles. After reconstructing the OPDs from these images, the integrated optical pathlength of each of the test objects was determined, and compared with the expected results. The parameter settings used in the reconstruction algorithm were found to be very important in obtaining a reliable measurement. Using optimal parameter settings, errors in the integrated OPD could be limited to a few per cent for circular objects within the investigated size range. For non-circular objects, however, the orientation angle of the object relative to the lateral shift was found to influence the measured values. Ellipses with their long axes perpendicular to the shift direction had a significantly higher integrated OPD than ellipses orientated parallel to the shift. By adjusting the reconstruction parameters the effect could be limited, but complete elimination of the artefact was not possible within the parameter range investigated.     

The three-dimensional point spread functions of a microscope objective in image and object space

The three-dimensional point spread function (3-D PSF) of an optical system in image space is distinguished from the 3-D PSF in object space and the relation between the two 3-D PSFs is derived. By using this relation one 3-D PSF can be easily obtained from the other. The 3-D PSFs are given in a single integral expression, which can be computed numerically. The results of this study can be used in 3-D image processing for microscopy and have been applied to the analysis of the diffusion of fluorescent molecules in a 3-D porous medium.